CN115521974A - SNP detection method and kit - Google Patents

Abstract

The invention discloses a SNP detection method and a kit. The formula includes: Tris, (NH4) ₂ SO ₄ , KCl, MgCl ₂ , dATP, dCTP, dGTP, dTTP, MTHFR 677R1, MTHFR 677T-F1, MTHFR C677-F1 , MTHFR 677T-MGB, MTHFR C677-MGB, Taq enzyme, DNA template and water, SNP detection method, comprising the following steps: Step 1, designing primers; Step 2, introducing mutations; Step 3, designing probes; Step 4, extracting Step 5, processing; Step 6, reaction; wherein in the above step one, ARMS qPCR primers are designed at the SNP position; wherein in the above step two, in order to reduce the non-specific amplification of primers, two allele primers are at the SNP position At 1-2 bases before and after the point, a mutation is artificially introduced; the positions of the introduced mutations are different; in the above step 3, an allele-specific MGB probe is designed; this invention develops a A new primer-probe design method for ARMS-qPCR, which solves the defect that ARMS-PCR cannot detect alleles in one tube.

Description

A kind of SNP detection method and kit

technical field

The invention relates to the technical field of biological detection, in particular to a SNP detection method and a kit.

Background technique

SNP is in the same group, there are two or more alleles at a certain locus, sometimes this allele difference will lead to large phenotype differences in individuals, in disease diagnosis, drug metabolism, disease It has high clinical value in terms of cause. The traditional SNP detection method generally uses the ARMS-PCR method, but because the ARMS-PCR is used to distinguish the mutation is designed on one of the primers, in the fluorescent PCR system, only two reaction wells can be used to compare the Ct value to distinguish; Therefore, it is necessary to design a SNP detection method and kit.

Contents of the invention

The purpose of the present invention is to provide a SNP detection method and a kit to solve the problems raised in the above-mentioned background technology.

To achieve the above object, the present invention provides the following technical solution: a SNP detection kit, the formula includes: Tris, (NH4) ₂ SO ₄ , KCl, MgCl ₂ , dATP, dCTP, dGTP, dTTP, MTHFR 677R1, MTHFR 677T- F1, MTHFRC677-F1, MTHFR 677T-MGB, MTHFR C677-MGB, Taq enzyme, DNA template and water, the dosage or final concentration of each component are: 10-50mM Tris, 125-375mM (NH4) ₂ SO _4. 0-50mM KCl, 1.5-5mM MgCl ₂ , 0.05-0.4uM dATP, 0.05-0.4uM dCTP, 0.05-0.4uM dGTP, 0.05-0.4uM dTTP, 0.05-1pM MTHFR 677R1, 0.05～0.5pM MTHFR 677T-F1, 0.05～0.5pM MTHFR C677-F1, 0.05～0.5pM MTHFR 677T-MGB, 0.05～0.5pM MTHFR C677-MGB, 0.05～0.2U/μl Taq enzyme, 1-50ng/reaction DNA template and appropriate amount of water.

Preferably, the Tris, (NH4) ₂ SO ₄ , KCl, MgCl ₂ , dATP, dCTP, dGTP, dTTP, MTHFR 677R1, MTHFR 677T-F1, MTHFR C677-F1, MTHFR 677T-MGB, MTHFR C677-MGB, The total volume of Taq enzyme, DNA template and water is 30 μL.

Preferably, the pH of the Tris is 8.3.

Preferably, the water is treated purified water.

A SNP detection method, comprising the following steps: Step 1, designing primers; Step 2, introducing mutations; Step 3, designing probes; Step 4, extracting; Step 5, processing; Step 6, reacting;

Wherein in the above step 1, ARMS-qPCR primers are designed at the SNP position;

In the above step 2, in order to reduce the non-specific amplification of the primers, the two allelic primers artificially introduce a mutation at 1-2 bases before and after the SNP site;

Wherein in the above-mentioned step 3, an allele-specific MGB probe is designed;

Wherein in the above step 4, use the peripheral blood DNA extraction kit to extract the EDTA or sodium citrate anticoagulated genomic DNA;

Among them, in the above step five, the SNP is processed by using a fluorescent PCR instrument, a high-speed centrifuge, a low-speed centrifuge, a constant temperature dry bath, a vortex oscillator, a refrigerator, and other equipment;

Among them, in the above-mentioned step 6, the SNP after processing in the step 5 is detected using a SNP detection kit, and in the process of detecting the reaction, the first step is to carry out a cycle of 10 minutes at an ambient temperature of 95°C; the second step In order to process at an ambient temperature of 95°C for 10 seconds, then at an ambient temperature of 60°C for 35 seconds, and then perform 45 cycles, set the fluorescence collection for detecting FAM and HEX at 60°C; the third step is to detect FAM and HEX at 25°C ℃ ambient temperature for 10 seconds; then the reaction results can be analyzed.

Preferably, in the second step, the positions of the introduced mutations are different.

Preferably, in the third step, there are 3 base differences in the alleles, so the MGB probe is not easy to develop color by non-specific hybridization.

Compared with the prior art, the beneficial effect of the present invention is: the invention develops a new primer probe design method for ARMS-qPCR, which solves the defect that ARMS-PCR cannot detect alleles in one tube; Not only the practicability of the invention is improved, but also the detection cost is reduced.

Description of drawings

Fig. 1-3 is the detection result analysis display figure of the present invention;

Fig. 4 is the design schematic diagram of the present invention;

Fig. 5 is a flow chart of the method of the present invention.

detailed description

The following will clearly and completely describe the technical solutions in the embodiments of the present invention with reference to the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only some, not all, embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by persons of ordinary skill in the art without making creative efforts belong to the protection scope of the present invention.

An embodiment provided by the invention:

Example 1:

A SNP detection kit, the formula includes: Tris, (NH4) ₂ SO ₄ , KCl, MgCl ₂ , dATP, dCTP, dGTP, dTTP, MTHFR 677R1, MTHFR 677T-F1, MTHFR C677-F1, MTHFR 677T-MGB, MTHFR C677-MGB, Taq enzyme, DNA template and water, the dosage or final concentration of each component are: 10mM Tris, 125mM (NH4) ₂ SO ₄ , 10mM KCl, 1.5mM MgCl ₂ , 0.05uM dATP, 0.05uM dCTP, 0.05uM dGTP, 0.05uM dTTP, 0.05pM MTHFR 677R1, 0.05pM MTHFR 677T-F1, 0.05pM MTHFR C677-F1, 0.05pM MTHFR677T-MGB, 0.05pM MTHFR C677-MGB, 0.05U/μl Taq enzyme, 1ng/reaction DNA template and appropriate amount of water; Tris, (NH4) ₂ SO ₄ , KCl, MgCl ₂ , dATP, dCTP, dGTP, dTTP, MTHFR 677R1, MTHFR 677T- The total volume of F1, MTHFRC677-F1, MTHFR 677T-MGB, MTHFR C677-MGB, Taq enzyme, DNA template and water is 30 μL; the pH of Tris is 8.3; the water is treated purified water.

Example 2:

A SNP detection kit, the formula includes: Tris, (NH4) ₂ SO ₄ , KCl, MgCl ₂ , dATP, dCTP, dGTP, dTTP, MTHFR 677R1, MTHFR 677T-F1, MTHFR C677-F1, MTHFR 677T-MGB, MTHFR C677-MGB, Taq enzyme, DNA template and water, the dosage or final concentration of each component are: 50mM Tris, 375mM (NH4) ₂ SO ₄ , 50mM KCl, 5mM MgCl ₂ , 0.4uM dATP , 0.4uM dCTP, 0.4uM dGTP, 0.4uM dTTP, 1pM MTHFR 677R1, 0.5pM MTHFR 677T-F1, 0.5pM MTHFR C677-F1, 0.5pM MTHFR 677T-MGB, 0.5pM MTHFR C677 -MGB, 0.2U/μl Taq enzyme, 50ng/reaction DNA template and appropriate amount of water; Tris, (NH4) ₂ SO ₄ , KCl, MgCl ₂ , dATP, dCTP, dGTP, dTTP, MTHFR 677R1, MTHFR 677T-F1 The total volume of , MTHFR C677-F1, MTHFR 677T-MGB, MTHFR C677-MGB, Taq enzyme, DNA template and water is 30 μL; the pH of Tris is 8.3; the water is treated purified water.

Example 3:

A SNP detection kit, the formula includes: Tris, (NH4) ₂ SO ₄ , KCl, MgCl ₂ , dATP, dCTP, dGTP, dTTP, MTHFR 677R1, MTHFR 677T-F1, MTHFR C677-F1, MTHFR 677T-MGB, MTHFR C677-MGB, Taq enzyme, DNA template and water, the dosage or final concentration of each component are: 30mM Tris, 200mM (NH4) ₂ SO ₄ , 20mM KCl, 3mM MgCl ₂ , 0.3uM dATP , 0.3uM dCTP, 0.3uM dGTP, 0.3uM dTTP, 0.3pM MTHFR 677R1, 0.3pM MTHFR 677T-F1, 0.3pM MTHFR C677-F1, 0.3pM MTHFR 677T-MGB, 0.3pM MTHFR C677-MGB, 0.1U/μl Taq enzyme, 30ng/reaction DNA template and appropriate amount of water; Tris, (NH4) ₂ SO ₄ , KCl, MgCl ₂ , dATP, dCTP, dGTP, dTTP, MTHFR 677R1, MTHFR 677T- The total volume of F1, MTHFR C677-F1, MTHFR 677T-MGB, MTHFR C677-MGB, Taq enzyme, DNA template and water is 30 μL; the pH of Tris is 8.3; the water is treated purified water.

Please refer to Figures 1-5, an embodiment provided by the present invention: a SNP detection method, comprising the following steps: Step 1, designing primers; Step 2, introducing mutations; Step 3, designing probes; Step 4, extracting; Step five, processing; Step six, reaction;

Wherein in the above step 1, ARMS-qPCR primers are designed at the SNP position in the MTHFR C677T gene;

In the above step 2, in order to reduce the non-specific amplification of the primers, the two allelic primers artificially introduce a mutation at 1-2 bases before and after the SNP site, and the positions of the introduced mutations are different. of;

Among them, in the above step 3, an allele-specific MGB probe is designed. Since there are 3 base differences in alleles, the MGB probe is not easy to develop color by non-specific hybridization; and it is used to detect the MTHFR C677T gene polymorphism The primer probe sequences are as follows:

Primer name sequence 5' mark 3' mark MTHFR677R1 AAGAATGTGTCAGCCTC none none MTHFR677T-F1 AGGTGTCTGCGGGcGt MTHFRC677-F1 AGGTGTCTGCGGGAac MTHFR677T-MGB TGATGAAATCGaCgC FAM MGB MTHFRC677-MGB TGATGAAATCGgtTC VIC MGB

MTHFR C677 gene sequence: AGGTGTCTGCGG GAGcCGATTTCATCA TCACGCAGCTTTCTTTGAGGCTGACACATTCTT; MTHFR 677T gene sequence: AGGTGTCTGCGG GAGtCGATTTCATCA TCACGCAGCTTTTCTTTGAGGCTGACACATTCTT;

Wherein in the above step 4, use the peripheral blood DNA extraction kit to extract the EDTA or sodium citrate anticoagulated genomic DNA;

Among them, in the above step five, the SNP is processed by using a fluorescent PCR instrument, a high-speed centrifuge, a low-speed centrifuge, a constant temperature dry bath, a vortex oscillator, a refrigerator, and other equipment;

Among them, in the above-mentioned step 6, the SNP after processing in the step 5 is detected using a SNP detection kit, and in the process of detecting the reaction, the first step is to carry out a cycle of 10 minutes at an ambient temperature of 95°C; the second step In order to process at an ambient temperature of 95°C for 10 seconds, then at an ambient temperature of 60°C for 35 seconds, and then perform 45 cycles, set the fluorescence collection for detecting FAM and HEX at 60°C; the third step is to collect the fluorescence at 25°C Process at ambient temperature of ℃ for 10 seconds; then the reaction results can be analyzed; and the analysis results are: C677 type result: ΔCt(FAM-VIC)>10, and the result display diagram is shown in Figure 1; 677T type result: ΔCt (FAM-VIC)<10, the results are shown in Figure 2; C677T heterozygous results: -2<ΔCt(FAM-VIC)<2, the results are shown in Figure 3.

Based on the above, the advantage of the present invention is that the present invention designs a new probe primer, changes the existing probe primer, and the probe primer set by the present invention can be used equally in the same detection tube. It is of high clinical value to detect the defect of bit gene.

It will be apparent to those skilled in the art that the invention is not limited to the details of the above-described exemplary embodiments, but that the invention can be embodied in other specific forms without departing from the spirit or essential characteristics of the invention. Accordingly, the embodiments should be regarded in all points of view as exemplary and not restrictive, the scope of the invention being defined by the appended claims rather than the foregoing description, and it is therefore intended that the scope of the invention be defined by the appended claims rather than by the foregoing description. All changes within the meaning and range of equivalents of the elements are embraced in the present invention. Any reference sign in a claim should not be construed as limiting the claim concerned.

Claims (7)

1. A SNP detection kit, the formula includes: Tris, (NH4) ₂ SO ₄ , KCl, MgCl ₂ , dATP, dCTP, dGTP, dTTP, MTHFR 677R1, MTHFR 677T-F1, MTHFR C677-F1, MTHFR 677T- MGB, MTHFR C677-MGB, Taq enzyme, DNA template and water, characterized in that: the amount or final concentration of each component is: 10-50mM Tris, 125-375mM (NH4) ₂ SO ₄ , 0-50mM KCl, 1.5~5mM MgCl ₂ , 0.05~0.4uM dATP, 0.05~0.4uM dCTP, 0.05~0.4uM dGTP, 0.05~0.4uM dTTP, 0.05~1pM MTHFR 677R1, 0.05~0.5pM MTHFR 677T-F1, 0.05～0.5pM MTHFR C677-F1, 0.05～0.5pM MTHFR 677T-MGB, 0.05～0.5pM MTHFR C677-MGB, 0.05～0.2U/μl Taq enzyme, 1～50ng/reaction DNA template and appropriate amount of water. 2. A SNP detection kit according to claim 1, characterized in that: said Tris, (NH4) ₂ SO ₄ , KCl, MgCl ₂ , dATP, dCTP, dGTP, dTTP, MTHFR 677R1, MTHFR 677T- The total volume of F1, MTHFR C677-F1, MTHFR 677T-MGB, MTHFR C677-MGB, Taq enzyme, DNA template, and water is 30 μL. 3. A kind of SNP detection kit according to claim 1, is characterized in that: the pH of described Tris is 8.3. 4. A kind of SNP detection kit according to claim 1, is characterized in that: described water is purified water through treatment. 5. A SNP detection method, comprising the following steps: Step 1, designing primers; Step 2, introducing mutations; Step 3, designing probes; Step 4, extracting; Step 5, processing; Step 6, reacting; It is characterized in that: Wherein in the above step 1, ARMS-qPCR primers are designed at the SNP position; In the above step 2, in order to reduce the non-specific amplification of the primers, the two allelic primers artificially introduce a mutation at 1-2 bases before and after the SNP site; Wherein in the above-mentioned step 3, an allele-specific MGB probe is designed; Wherein in the above step 4, use the peripheral blood DNA extraction kit to extract the EDTA or sodium citrate anticoagulated genomic DNA; Among them, in the above-mentioned step five, the SNP is processed by using a fluorescent PCR instrument, a high-speed centrifuge, a low-speed centrifuge, a constant temperature dry bath, a vortex shaker, a refrigerator, and other equipment; Among them, in the above-mentioned step 6, the SNP after processing in the step 5 is detected using a SNP detection kit, and in the process of detecting the reaction, the first step is to carry out a cycle of 10 minutes at an ambient temperature of 95°C; the second step In order to process at an ambient temperature of 95°C for 10 seconds, then at an ambient temperature of 60°C for 35 seconds, and then perform 45 cycles, set the fluorescence collection for detecting FAM and HEX at 60°C; the third step is to detect FAM and HEX at 25°C ℃ ambient temperature for 10 seconds; then the reaction results can be analyzed. 6. A SNP detection method according to claim 5, characterized in that: in said step 2, the positions of the introduced mutations are different. 7. A SNP detection method according to claim 5, characterized in that: in said step 3, there are 3 base differences in alleles, so the MGB probe is not easy to develop color by non-specific hybridization.

Images